The AAA (ATPase associated with a variety of activities) ATPase p97 is conserved across all eukaryotes and is essential for life in budding yeast (Giaever, G., et. al. Nature (2002) 418, 387-391) and mice (Muller, J. M. et al. Biochem. Biophys. Res. Commun. (2007) 354, 459-465). Humans bearing reduction-of-function alleles of p97 are afflicted with a syndrome that includes inclusion body myopathy and frontotemporal lobar degeneration (Weihl, C. et al. Hum. Mol. Genet. (2006) 15, 189-199). Loss-of-function studies in model organisms indicate that p97 plays a critical role in a broad array of cellular processes including Golgi membrane reassembly (Rabouille, C. et al. Cell (1995) 82, 905-914), membrane transport (Ye, Y. et al Nature (2001) 414, 652-656; Ye, Y. et al. Nature (2004) 429, 841-847) degradation of misfolded membrane and secretory proteins by the ubiquitin-proteasome system (UPS) (Golbik, R. et al. Biol. Chem. (1999) 380, 1049-1062; Richly, H. et al. Cell (2005) 120, 73-84), regulation of myofibril assembly (Janiesch, P. C. et al. Nat. Cell Biol. (2007) 9, 379-390), and cell division (Cao, K. et al. Cell (2003) 115, 355-367). The broad range of cellular functions for this protein are thought to derive from its ability to unfold proteins or disassemble protein complexes. The mechanochemical activity of p97 is linked to substrate proteins by an array of at least 14 UBX domain adapters that bind p97, as well as the non-UBX domain adaptors Ufd1 and Npl4 (Meyer, H. H. et al. EMBO J. (2000) 19, 2181-2192).
The sequence of p97 reveals three domains (N-domain, D1 ATPase domain, and D2 ATPase domain) joined by linker regions. X-ray crystallography of p97 revealed that it forms a homohexamer of 97 kilodalton subunits that assemble to form two stacked rings. The two rings are formed by the ATPase domains (Huyton, T. et al. Jan. 16, 2009. Struct. Biol. (2003) 144, 337-348; DeLaBarre, B. et al. Nat. Struct. Biol. (2003) 10, 856-863). The ‘top’ ring is formed by a hexamer of the D1 domains, whereas the ‘bottom’ ring is formed by a hexamer of the D2 domains. The N-domain extends outward from the D1 domain ring. Although it is clear that the D2 domain hydrolyzes ATP in vitro, the level of D1-specific ATPase activity reported by different investigators varies. Nevertheless, genetic studies in yeast suggest that ATP hydrolysis by both the D1 and D2 domains is essential for the function of p97 (Song, C. et al. J. Biol. Chem. (2003) 278, 3648-3655; Ye, Y. et al. J. Cell Biol. (2004) 162, 71-84). Binding of ATP to the D1 domain is also required for assembly of p97 (Wang, Q. et al. Biochem. Biophys. Res. Commun. (2003) 300, 253-260). Although ATP hydrolysis by the D2 domain is not required for assembly of p97 hexamer, it is thought that ATP hydrolysis by the D2 domain is an obligate step in the catalytic cycle of p97, and contributes to structural transformations in bound substrates, resulting in their unfolding or dissociation from bound partners.
A prominent cellular function for p97 that has received considerable scrutiny is its role in the turnover of misfolded secretory proteins via the UPS. In this process, which is known as ERAD (for endoplasmic reticulum-associated degradation), proteins that fail to fold within the ER are retrotranslocated in a p97-dependent manner into the cytoplasm where they are degraded by the UPS (Ye, Y. et al. Nature (2004) 429, 841-847). In this process, p97 is thought to mediate extraction of substrates from the ER membrane. p97 is also required for the turnover of cytosolic substrates of the UPS (Janiesch, P. C. et al. Nat. Cell Biol. (2007) 9, 379-390; Cao, K. et al. Cell (2003) 115, 355-367; Fu, X. et al. J. Cell Biol. (2003) 163, 21-26), although its role in turnover of cytosolic proteins is less understood.
p97 represents a suitable target for for cancer therapeutics. p97 is essential, and so drugs that inhibit it should be antiproliferative. Also, p97 is known to be overproduced in multiple cancers (Yamamoto, S. et al. Ann. Surg. Oncol. (2005) 12, 925-934; Yamamoto, S. et al. Clin. Cancer Res. (2004) 10, 5558-5565; Yamamoto, S. et al. Ann. Surg. Oncol. (2004) 11, 697-704; Yamamoto, S. et al. Ann. Surg. Oncol. (2004) 11, 165-172) suggesting that its activity may be rate-limiting for the development of at least some cancers. p97 is known to be essential for ERAD (Carvalho, P. et al. Cell (2006) 126, 361-373), and recent studies suggest that cancer cells may be particularly dependent upon ERAD (Boelens, J. et al. In Vivo (2007) 21, 215-226). Furthermore, p97 has been linked to the turnover of IkB and consequent activation of NF-kB (Dai, R. M. et al. J. Biol. Chem. (1998) 273, 3562-3573). NF-kB activity is important for the survival of some tumor cells, particularly in multiple myeloma (Keats, J. J. et. al. Cancer Cell (2007) 12, 131-144; Annunziata, C. M. et. al. Cancer Cell (2007) 12, 115-130). It has been suggested that bortezomib is active in multiple myeloma due to its ability to block turnover of proteins via the ERAD pathway and its ability to block turnover of IkB, thereby squelching the activity of NF-kB. Given that p97 is implicated in both ERAD and IkB turnover but otherwise has a more restricted role in the UPS compared to the proteasome itself, drugs that target p97 may retain much of the efficacy of bortezomib but with less toxicity.
Thus there exists a need in the art for compounds for and methods of inhibiting the activity of p97.